* Each gene you analyze requires a set of three oligos: '''forward primer''', '''reverse primer''', and a '''UPL probe'''.

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* Use Roche's [http://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=UP030000 Assay Design Center] to design optimal primers and identify the right probe for your gene(s) of interest.

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* Use Roche's [http://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=UP030000 Assay Design Center] to design optimal primers, and to identify the right UPL probe for your gene(s) of interest.

* The forward and reverse primers need to be ordered from a DNA synthesis company (e.g., IDT DNA, Promega, etc.), and the UPL oligo comes from Roche.

* The forward and reverse primers need to be ordered from a DNA synthesis company (e.g., IDT DNA, Promega, etc.), and the UPL oligo comes from Roche.

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2. '''Design your reactions'''<br>

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* ''How many reactions should I plan to run?'' Each experimental cDNA sample is a '''template'''. The gene being detected is often referred to as a '''target'''. You should also include a '''reference''' target gene (a housekeeping gene that is always active, not expected to change). Each unique template and target combination requires its own reaction. You will also need to set up a '''no template control''' to observe the amount of background noise from that reaction.

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* Hypothetical example: A scientist wants to measure differences the expression of genes A, B, and C in an experiment where cells were treated with a drug, or untreated. She will use GAPD as the reference gene, using the Roche-supplied primer set. All of the unique reactions she must set up are:<br>

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'''REACTION LIST'''

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2. '''Set up a reaction list and plan the plate layout'''<br>

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{| class="wikitable" style="width: 400px; height: 200px;"

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* ''How many reactions should I plan to run?'' Each experimental cDNA sample is a '''Template'''. The gene being detected is often referred to as a '''Gene Target'''. You should also include a '''reference''' Gene Target such as GAPD (a housekeeping gene that is always active, not expected to change). Each unique template and target combination requires its own reaction. You will also need to set up a '''no template control''' to observe the amount of background noise from that reaction.

Both Variations (1 and 2) are correct. Choose a format that helps you to easily keep track of the samples. If you have a large experiment, try to fit as many reactions on the well as you can (to avoid wasting plates), but also keep the samples arranged in an orderly fashion so that the set-up won't confuse you.

This hypothetical experiment requires '''12 Rxns x 3 replicates = <u>36 wells</u>'''. If you need more than 96 wells, you must split the experiment over multiple plates. This plate is set up so that there is one template per row, and a target for every three columns. You can use whatever organization suits your experiment. It is absolutely critical that you keep a '''reaction list''' and '''plate layout''' in your notes. Your plate set-up will probably vary for each run.

* If you need more than 96 wells, you must split the experiment over multiple plates.

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* It is absolutely critical that you keep a '''reaction list''' and '''plate layout''' in your notes. Your plate set-up will probably vary for each run.

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3. '''Reaction Set-up: PCR master mixes for each gene target'''<br>

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* Create a PCR master mix for every unique primer set.

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* In the example above, primer set A is needed for 3 unique reactions, with 3 technical replicates each. Thus, enough master mix should be made for '''3 Rxns x 3 replicates + 1 extra = <u>10 individual wells</u>''' (the "extra" is included so that you don't run out of master mix). The same needs to be done for primer sets B, C, and D in separate tubes (each column in the table below is a 1.5 mL tube).

* Typically, you will have only 20 μL of stock cDNA on hand. You use a little of the stock cDNA to make a separate dilution of cDNA to extend its use. For many reactions, a 1:10 dilution is suitable. For GAPDH, you should use a 1:1000 or 1:10,000 dilution since this gene is expressed at levels so high, it can produce saturating qPCR signals

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* '''MPK14''' - 85.0 μL

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* In the example above, treated cell cDNA is needed for 4 unique reactions, with 3 technical replicates each. Thus, enough master mix should be made for '''4 Rxns x 3 replicates + 1 extra = <u>13 individual wells</u>''' (the "extra" is included so that you don't run out of master mix). The same needs to be done for templates "untreated" and "no template" in separate tubes (each column in the table below is a 1.5 mL tube).

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* '''CBX8''' - 85.0 μL

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* '''TNF''' - 85.0 μL

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* '''NPPA''' - 85.0 μL

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* '''GAPD''' - 85.0 μL

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4. '''Reaction set-up: master mixes for each Template'''<br>

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* Typically, you will have only 20 μL of stock cDNA on hand.

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* Make a 1:10 dilution of cDNA by adding 10 μL of the stock cDNA to 90 μL of PCR H<sub>2</sub>O.

Wet bench: Add the appropriate combinations of of the master mixes to the 96-well plate

---> Run the reaction in the Light Cycler!

1. Design your primers

Each gene you analyze requires a set of three oligos: forward primer, reverse primer, and a UPL probe.

Use Roche's Assay Design Center to design optimal primers, and to identify the right UPL probe for your gene(s) of interest.

The forward and reverse primers need to be ordered from a DNA synthesis company (e.g., IDT DNA, Promega, etc.), and the UPL oligo comes from Roche.

2. Set up a reaction list and plan the plate layout

How many reactions should I plan to run? Each experimental cDNA sample is a Template. The gene being detected is often referred to as a Gene Target. You should also include a reference Gene Target such as GAPD (a housekeeping gene that is always active, not expected to change). Each unique template and target combination requires its own reaction. You will also need to set up a no template control to observe the amount of background noise from that reaction.

Hypothetical example:

cDNA samples + a no template control = 3

Gene targets + GAPD reference = 5

Replicates per reaction = 3

Wells needed = 3 * 5 * 3 = 45

REACTION LIST

Template cDNA

Gene Target

Rxn 1:

treated cells

MPK14

Rxn 2:

treated cells

CBX8

Rxn 3:

treated cells

TNF

Rxn 4:

treated cells

NPPA

Rxn 5:

treated cells

GAPD (reference gene)

Rxn 6:

untreated cells

MPK14

Rxn 7:

untreated cells

CBX8

Rxn 8:

untreated cells

TNF

Rxn 9:

untreated cells

NPPA

Rxn 10:

untreated cells

GAPD (reference gene)

Rxn 11:

no template

MPK14

Rxn 12:

no template

CBX8

Rxn 13:

no template

TNF

Rxn 14:

no template

NPPA

Rxn 15:

no template

GAPD (reference gene)

PLATE LAYOUT

Variation 1
Variation 2
Both Variations (1 and 2) are correct. Choose a format that helps you to easily keep track of the samples. If you have a large experiment, try to fit as many reactions on the well as you can (to avoid wasting plates), but also keep the samples arranged in an orderly fashion so that the set-up won't confuse you.

A single plate contains 96 wells. To insure accuracy, three technical replicates per reaction (Rxn) are required

If you need more than 96 wells, you must split the experiment over multiple plates.

It is absolutely critical that you keep a reaction list and plate layout in your notes. Your plate set-up will probably vary for each run.

3. Reaction set-up: PCR master mixes for each Gene Target

Label one 1.5 mL tube per gene target

Make enough PCR master mix for your plate...

MPK14 is in Reactions 1, 6, and 11 = 3

Replicates per reaction = 3

Master mix amount = 3 * 3 + 1 (to allow for pipetting error) = 10

The same needs to be done for CBX8, TNF, NPPA, and GAPD in separate tubes.